The reaction catalyzed by mitochondrial pyruvate dehydrogenase complex (PDC) links glycolysis and Citric Acid Cycle. However, physiological significance of this reaction goes beyond its role in energy production because it also serves as a source of carbon for biosynthesis of sterols and fatty acid and provides the general means to control the tissue levels of pyruvate. Consequently, this reaction plays an important role in general metabolism, in adaptation to starvation and hypoxia, in diabetes, ischemia, and cancer. Mammalian PDC is regulated through the reversible phosphorylation (inactivation)/dephosphorylation (re-activation) cycle catalyzed by multiple isozymes of pyruvate dehydrogenase kinase (PDHK1, PDHK2, PDHK3, and PDHK4) and pyruvate dehydrogenase phosphatase (PDP1 and PDP2). The reversible phosphorylation accounts for the short- and long-term regulation of PDC. Within the past decade, significant progress has been made in structural and biochemical characterization of PDHK and PDP isozymes. On the other hand, their roles in regulation of PDC in starvation, diabetes, or cancer remain poorly understood. It is generally believed that, at least in starvation and diabetes, the long-term regulation of PDC largely reflects the induction of isozyme PDHK4. However, the lack of a clear phenotype in PDHK4-/- mouse model strongly suggests the existence of alternative mechanism(s). Our preliminary data indicate that PDHK2 might be crucial for regulation of PDC in starvation, while PDHK3 might contribute to the Warburg effect in cancer cells. In this application, we propose to explore these hypotheses using PDHK2-/-, PDHK3-/-, and PDHK4-/- knockout mouse models. This will be achieved through the following Specific Aims: 1) to identify the molecular basis of stable changes in the specific activity of PDHK2; 2) to elucidate the role of PDHK2 in adaptation to starvation; and 3) to establish the physiological role of PDHK3 in regulation of PDC. Accomplishment of these objectives will shed new light on the role of pyruvate dehydrogenase reaction in general carbohydrate and lipid metabolism, in adaptation to starvation, and in cancer. In the long run, it may lead to the development of highly specific drugs that will alleviate complications associated with diabetes, ischemia, and cancer. PUBLIC HEALTH RELEVANCE: The long-term regulation of PDHK activity is central to adaptation to food deprivation, hypoxia, high-fat diet, etc. However, when similar mechanisms are activated in diabetes, ischemia, or cancer, the outcomes are detrimental. Thus, uncovering the molecular mechanisms responsible for the regulation of PDHK is crucial for the development of new therapeuticals.